1. Field of the Invention
In general, this invention relates to an antithrombin III-independent, heparin cofactor II (hereinafter HCII) mediated inhibition of thrombin generation, as well as the inhibition of the alternative, classical and terminal pathways of complement activation; specifically, this invention relates to a chemically-sulfated dermatan sulfate composed of mainly disulfated disaccharide dermatan chains which inhibit thrombin generation and complement activation.
2. State of the Art
Conditions or diseases characterized by excessive generation of thrombin, such as deep vein thrombosis (hereinafter DVT) or destructive vascular smooth muscle cell (hereinafter VSMC) proliferation can be life threatening and require effective treatment. These conditions frequently occur after the subject has been exposed to trauma, such as that caused by surgery or other wounds. The trauma results both in vascular damage and VSMC proliferation, causing vascular stenosis, restenosis and hyperplasia, as well as activation of blood coagulation. Vascular restenosis, hyperplasia and activation of blood coagulation can be life threatening when occurring after vascular graft surgery, heart transplantation, balloon or laser angioplasty, arterial traumatic injury, post-surgical repair of muscular arteries, long term in-dwelling of arterial catheters, invasive arterial diagnostic procedures, kidney, lung or liver transplants, or bypass surgery procedures.
DVT, for example, frequently occurs before one life threatening complication, pulmonary embolism (hereinafter PE). The majority of PEs, and particularly, the majority of fatal PEs, follow an asymptomatic DVT. Epidemiological data indicate the rate of DVT each year to be about 160 per 100,000 in the general population, with the rate of fatal PEs about 60 per 100,000. Thus, the medical profession strives to treat DVT and prevent its complications. Ideally, a satisfactory treatment of DVT will prevent PE, extension of the thrombus, venous gangrene and limb losses, symptomatic recurrence of thrombosis, severe post-thrombotic syndrome, and progressive swelling of the leg resulting in increased compartmental pressure leading to subsequent phlegmasia cerulea dolens.
As an example of the seriousness of the problem, patients undergoing some sorts of elective surgery are at high risk of developing postoperative venous thromboembolism. It is known that without preoperative prophylaxis a high incidence, perhaps as high as 50%, of DVT accompanies total hip replacement with a consequent high rate of PE. However, anticoagulants are efficacious for the prevention of morbidity and mortality in the treatment of DVT (European Consensus Report; 1992) since it is known that anticoagulants, in general, and heparin in particular, can reduce DVT and fatal PE in general surgical patients. Heparin, a glycosaminoglycan (hereinafter GAG) having potent anticoagulant properties, is a heterogeneous mixture of variably sulfated polysaccharide chains having a molecular weight between about 5,000 to about 30,000 Daltons composed of repeating units of D-glucosamine and either L-iduronic or D-glucuronic acid residues. Therefore, prudent procedure dictates that before surgical intervention the patient receives anticoagulant prophylaxis, usually heparin, which is typically continued for 7 to 10 days postoperatively.
Other life threatening complications occur after surgical intervention to repair vascular damage. One serious form of vascular damage, atherosclerosis, causes nearly 50% of all mortality in North America from a diverse array of illness including heart attack, stroke and gangrene of the extremities. Usually, an excessive, inflammatory-fibroproliferative response to various forms of endothelium and arterial wall insult causes the atherosclerotic lesions. Frequently, the treatment of choice to combat atherosclerosis is surgical intervention, resulting in the over 1.5 million bypass graft, endarterectomy, and percutaneous translumenal angioplasty (hereinafter PCTA) procedures performed annually in North America. Unfortunately, in many instances, although the immediate effects of the procedure are beneficial to the surgical patient, later post-procedure acceleration of the arteriosclerotic process greatly reduces the long-term effectiveness of the surgical intervention. Specifically, VSMC proliferation in the intima frequently leads to stenosis and occlusion of the lumen of the vessel. For example, the restenosis rate following PCTA is as high as 40% in the first three to six months after the procedure. In the case of the over 200,000 bypass grafts performed annually, between 40 to 60% fail within five years due to proliferative and occlusive changes involving VSMC proliferation. Moreover, VSMC proliferation accounts for more than 50% of heart transplant failures within five years. Accordingly, there is a need for compositions and methods to reduce the acceleration of arteriolsclerotic processes occurring after cardiovascular procedures.
Drugs or other treatments interfering with the growth-promoting activity causing VSMC proliferation would slow progression of the atherosclerotic process. Since it is known that thrombin exerts a potent VSMC growth promoting activity, and, under certain conditions may be present within the vessel wall, thrombin inhibitors are prime candidates to slow the atherosclerotic process. Among the compositions that interfere with the effect of thrombin are commercial heparins, (Castellot, J. J. J. Cell Biol. 102: 1979-84 (1986)) which inhibit thrombin both in vitro and in vivo. Heparin is also thought to be a potent anti-proliferative inhibiting thrombin better than any of the other known GAG in vitro. (Castellot, J. J., et al. J. Cell Biol. 90: 3722 (1981)) However, there is little evidence to support the latter possibility in the clinical setting.
Moreover, heparin has many limitations caused primarily by its potent anticoagulant activity. For example, the high doses of heparin (&gt;200 units/kg (hereinafter U/kg), which generates &gt;3 antithrombin units/ml of plasma) required to perform successful cardiopulmonary bypass (hereinafter CPB) surgery and to maintain CPB pump patency causes local or profuse hemorrhage contributing to patient morbidity. Excessive post-operative blood loss and the consequent requirement for blood transfusion are well-documented adverse affects suffered by patients who undergo CPB surgery. (Woodman, R. D., Harker, L. A., Bleeding Complications Associated with Cardiopulmonary Bypass, Blood 76(9): 1680 (1990); Dietrich, W., et al., The Influence of Preoperative Anticoagulation on Heparin Response During Cardiopulmonary Bypass, J. Thorac. Cardiovasc. Surg. 102: 505 (1991)) Life threatening bleeding is reported in between 5 to 25% of cases and it is estimated that surgeons must reopen approximately three percent of CPB patients because of surgical bleeding. The bleeding complications are due, not only to surgical bleeds, but also to a displacement of heparin from various plasma proteins which results in prolonged systemic anticoagulation and to an inhibitory effect of heparin on platelet function. Administering a precise amount of heparin antagonist in the form of protamine reverses the anticoagulant effect of heparin and prevents excessive blood loss at the time of decannulation. However, protamine administration is critically dosage sensitive, and even modest excess protamine administration causes numerous alarming and potentially fatal side-effects, including platelet inhibition, prolongation of the activated partial thromboplastin time (hereinafter APTT), and prolongation of systemic hypotension and pulmonary hypertension. (Ireland, H., Rylance, P. B., Kesteven, P., Heparin as an Anticoagulant During Extracorporeal Circulation, Heparin, David A. Lane and Ulf Lindahl (Eds).; 549-74 (1989)) A 1985 survey of perfusionists cited "the protamine reaction" as the most frequent perfusion accident in CPB procedures, observed fully in two-thirds of the cases. (Kuruz, 6th Annual Meeting of Pathophysiology and Extracorporeal Technology, San Diego, Calif. (1986)) Therefore, CPB provides an excellent in vivo model to evaluate the contribution candidate compounds or methods have on the exacerbation or the amelioration of antithrombotic activity and bleeding complications.
Heparin has also been used to treat complement system abnormalities. The complement system, plays a major role in host defense both through destruction of invading organisms and through mediation of inflammation. Complement abnormalities are unusual conditions characterized by a deficiency or by a dysfunction of any of the more than nineteen normally well-behaved proteins constituting about 10% of the globulins in normal human serum. Patients with complement deficiencies or with complementary dysfunctions also may be susceptible to tissue injury as a result of excessive inflammatory responses. Further, complement activation in the course of recovery from temporary blood vessel occlusion or in response to cardiopulmonary bypass during heart surgery initiates tissue damage beyond that caused by the initial injury. Heparin has been shown to inhibit activity of the alternative, classical and terminal pathways of complement by regulating C1, C1 Inhibitor, C4 binding protein, C3b, factor H and S-protein in a model that predicts in vivo complement inhibition properties. (Edens, R. E., Linhardt, R. J., Bell, C. S., Weiler, J. M., Heparin and Derivatized Heparin Inhibit Zymosan and Cobra Venom Factor Activation of Complement in Serum, Immunopharmacol. 27: 145153 (1994)) However, the anticoagulant activity of heparin contributes to an increased risk of bleeding, electrolyte shifts and thrombocytopenia.
In an effort to counteract thrombus formation and bleeding caused by the systemic administration of heparin during CPB surgery, methods for site-specific administration of heparin have been developed involving coating antithrombotic compounds on blood-interacting biomaterials. The antithrombotic compound may be bound either ionically or covalently onto a biomaterial polymer surface. A major disadvantage with polymers coated with currently available heparin formulations is the limited efficacy of heparin. Therefore, it is important to develop new coating compositions which optimize antithrombotic activity while being applied satisfactorily and consistently to a variety of materials such as natural polymers and synthetic plastics. Such new compositions will, ideally, result in complete coverage of the blood-interacting surfaces of a medical article.
Heparin is a natural product, and, as is the case with many products obtained from different biological sources, heparin can vary in its structure, particularly in the degree of sulfation. Selective O-sulfation enhances the activity of some heparins and heparin-like GAGs. Whale heparin, for example, having a low degree of O-sulfation, can be selectively 6-O-sulfated as the tributylammonium salt in dimethylformamide (Uchiyama, H., Metori, A., Ogamo, A., Nagasawa, K. J. Biochem. 107: 377 (1990); Ogamo, A., Metori, A., Uchiyama, H., Hagasawa, K. Carboh. Res. 193: 165-172 (1989)) to increase its anticoagulant activity. Pyridium salts of heparan sulfate, another GAG with a low level of N- and O-sulfation, have been sulfated (Ofosu, F. A., Modi, G. J., Blachman, M. A., Buchanan, M. R., Johnson, E. A. Biochem. J. 248: 889 (1987)) to increase biological activity. Alternatively, GAGs, being soluble in water or formamide (Kiss, J. Helv. Chimica. Acta. 50: 1423 (1967); Griffin, C. C., Stevenson, J. R., Foley, K. M. XIVth Int. Carbohydr. Symp., Stockholm (1988)), have also been sulfated by treatment with pyridine or trialkyl amines sulfur trioxides complexes, the latter being preferred because of its stability. (Levy, L., Petacek, F. J. Proc. Soc. Exp. Biol. Med.109: 901 (1962)) Increasing the degree of sulfation improves the catalytic effects of dermatan sulfate (hereinafter DS) on the inhibition of thrombin by HCII in plasma. (Ofosu, F. A., Modi, G. J., Smith, L. M., Cerskus, A. L., Hirsch, J., Blajchman, M. A. Blood 64: 742-47 (1984)) Other studies have suggested a potential as antithrombotic drugs for slightly over-sulfated derivatives of dermatan sulfate. (Maaroufi, R. M., Tapon Bretaddiere, J., Mardiguian, J., Sternberg, C., Dautzenberg, M. D., Fischer, A. M., Influence of the Oversulfation Method and the Degree of Sulfation on the Anticoagulant Properties of Dermatan Sulfate Derivatives. Thromb. Res. 59: 749-758 (1990)) However, none of these over-sulfated GAGs have been shown to overcome the difficulties noted above for heparin in the clinical setting.
Recent studies provide other rationales for replacing heparin as a thrombin inhibitor. For example, thrombin may bind to fibrin in vitro. This binding significantly impairs the ability of heparin to catalyze thrombin inhibition (Hogg, D. J., Jackson, C. M., Fibrin Monomer Protects Thrombin from Inactivation by Heparin antithrombin III: Implications for Heparin Therapy, Proc. Natl. Acad. Sci. 86: 3619-238 (1989)) because heparin must bind both to the high affinity site on ATIII, and to the thrombin anionic exosite--the same site at which thrombin binds fibrin--to catalyze thrombin inhibition. Therefore, thrombin bound to fibrin impairs the access of the exosite to heparin/ATIII, greatly reducing the effect of heparin as a thrombin inhibitor.
Use of heparin allows complex vascular surgical procedures. However, as can be seen from the above discussion, heparin, particularly if administered systemically, is not the ideal candidate drug for inhibition of thrombosis, the prevention of accelerated arteriosclerosis involving VSMC growth, or inhibition of complement activation that accompanies vascular procedures and organ transplants.
One promising replacement candidate for heparin is dermatan sulfate, a heparin-like GAG, also known as .beta.-heparin or chondroitin sulfate B. Dermatan sulfate is a polysaccharide composed of repeating uronic acid-&gt;N-acetyl-D-galactosamine disaccharides joined by alternating 1,3 and 1,4 linkages. Depending on its source and the method of preparation, it can have a molecular weight as high as 50,000 Daltons. Initially, it is formed as a polymer composed of repeating uronosyl-&gt;N-acetyl-D-galactosamine disaccharide units attached to the core protein via a glucuronosyl-&gt;galactosyl-&gt;galactosyl-&gt;xylosyl linkage region. Since the monomeric components of dermatan sulfate are saccharides, they are susceptible to epimerization. In particular, in the biosynthesis of dermatan sulfate, some of the D-glucuronic acid residues are epimerized at C-5, converting them to L-iduronic acid residues, followed by O-sulfation of N-acetyl-D-galactosamine primarily at C-4, but also at C-6. Dermatan sulfate typically has sulfur and nitrogen contents between about 6.2 to 6.9% and between about 2.4 to 2.9%, respectively (Seikagaku America, Inc. 3 (1989)), which structurally reflects a dermatan monosulfated disaccharide, referred to herein as dermatan sulfate (DS).
It has been shown that dermatan sulfate and chondroitin sulfate components in cartilage are solely responsible for the cation-binding capacity, that the cation-binding reactions are of the ion-exchange type and that different cations exhibit varying degrees of affinity for the cartilage. (Dunstone JR, Ion-Exchange Reactions Between Acid Mucopolysaccharides and Various Cations. Biochem J. 85: 336-351 (1962))
In animal models, DS has been demonstrated to be a potent antithrombotic agent with low risk of hemorrhage. (Fernandez, F., Van Rijn, J., Ofosu F. A., Buchanan, M. R., The Hemorrhagic and Anthrombotic Effects of Dermatan Sulfate, Brit. J. Haematol. 64: 3 (1986)) A 500 .mu.g/kg dose of DS inhibits thrombus formation as well as a 70 .mu.g/kg dose of heparin. At higher doses, the amount of bleeding from a standardized incision in a rabbit's ear was much greater with heparin than with DS, indicating that DS is more efficient and safer as an anticoagulant than heparin, suggesting that DS would be useful clinically to inhibit thrombus formation without triggering massive hemorrhage. In addition, DS has little in vitro effect on platelets, and one may inject up to forty times the antithrombotic dose of DS in rabbits without increasing bleeding. (Fernandez, F., Van Rijn, J. Ofosu, F. A., Hirsch, J., Buchanan, M. R., The hemorrhagic and antithrombotic effect of dermatan sulfate. Br. J. Haematol. 64: 309-1 (1986)) Moreover, DS has been shown to effectively catalyze the inhibition of thrombin in vitro, regardless of whether it is fibrin-bound or free, (Okwusidi, J. I., Anvari, N., Kulczycky, M., Blajchman, M. A., Buchanan, M. R., Ofosu, F. A., In vivo Catalysis of Thrombin Inhibition by Antithrombin III or Heparin Co-factor II and Antithrombotic Effect: Differential Effects of Unfractionated Heparin and Dermatan Sulfate, Thomb. Haemorrh. Disorders. 1: 77-8013 (1990)), and to inhibit both thrombin and fibrin accretion onto preformed rabbit thrombi more effectively than heparin in vivo. DS (30 U/kg) has also been shown to inhibit hyperplasia of 1st and 2nd injury carotid arteries in a rabbit model in which heparin (150 U/kg) had no effect. (Buchanan M. R., Brister S. J. Inhibition of Injured Vessel Wall Restenosis with Acute Thrombin Inhibition. Relative Effects Of Heparin and Dermatan Sulfate. Bk of Abst. Joint Conf Arteriosclerosis, Thrombosis, Vascular Biol, Salt Lake City, Utah. p. 18 (Feb. 18-20, 1987)).
DS specifically activates HCII, a plasma protease inhibitor, which inhibits thrombin but not other proteases involved in hemostasis. (Tollefsen, D. W., Majerus, D. W., Blank, M. K., Heparin Cofactor II. Purification and Properties of a Heparin--Dependent Inhibitor of Thrombin in Human Plasma, J. Biol. Chem. 257:2162-9 (1982)) HCII is activated by DS fractions of 12 or more residues in length that contain an octasaccharide sequence required for binding to the inhibitor. A hexasaccharide component in DS with high affinity to HCII has been identified. (Tollefsen, D. M., In: Lane, D. A., Bjurk, I., Lindahl, U (Eds.), Heparin and Related Polysaccharides. Plenum Press, New York, pp. 167-7 (1992)).
DS inhibits thrombin through an ATIII-independent pathway by accelerating HCII-dependent inhibition of thrombin. (Ofosu, F. A., Modi, G. J., Blachman, M. A., Buchanan, M. R., Johnson, E. A. Biochem. J. 248: 889 (1987)) DS contains some amount of oversulfated sequences (IdoA2S-GalNAc4S) and (IdoA-GalNAc4S6S) besides the major monosulfated disaccharide sequence (IdoA-GalNAc4S). The concentration of the oversulfated sequences in naturally occurring DS correlates with the HCII-mediated inhibition of thrombin. (Mascellani, G., Liverani, L., Prete, A., Guppola, P. A., Bergonzini, G., Bianchini, P., Relative Influence of Different Disulphate Disaccharide Clusters on the HCII-mediated Inhibition of Thrombin by Dermatan Sulfates of Different Origins, Thromb. Res. 74: 605-1517 (1994)) It has been suggested that the L-iduronic acid content in DS correlates with increased thrombin inhibition. (Whinna H. C., Choi H. U., Rosenberg L. C., Church F. C. Interaction of Heparin Cofactor II with Biglycan and Decorin, J. Biol Chem. 268: 3920-3924 (1993) DS has less specific anticoagulant activity than heparin, as demonstrated by comparing their activities. Compared to heparin 150 U/mg, DS has an activity of less than 5 U/mg, as measured by the APTT. (Thomas, D. P., Merton, R. E., Barrowcliffe, T. W., Relative Efficacy of Heparin and Related GAGs as Antithrombotic Drugs, Ann. N.Y. Acad. Sci. 556: 313-22 (1989)) DS has been demonstrated to be an effective anticoagulant for DVT prophylaxis in patients undergoing elective orthopedic surgery, for preventing thrombus formation in patients undergoing hemodialysis, and for performing successful cardiopulmonary bypass in adult pigs experimentally. (Van Rijn, J., McKenna, J., Dermatan Sulfate: A New Concept in Antithrombotic Therapy, Diss. Abstr. Int. B 53: 5662 (1993)); (Ryan, K. E., Lane, D. A., Flynn, A., Ireland, H., Boisclair, M., Sheppard. J., Curtis, J. R., Antithrombotic Properties of Dermatan Sulfate (MF701) in Haemodialysis for Chronic Renal Failure. Thom Haemostas 68: 563 (1992); (Brister S. J., Ofosu, F. A., Heigenhauser G. L. F., Gianese, F., Buchanan M. R., Is Heparin the Ideal Anticoagulant for Cardiopulmonary Bypass? Dermatan Sulphate May Be ab Alternate Choice. Thom Haemostas 71: 468-73 (1994)).
Unfortunately, however effective DS is in the laboratory, it causes practical clinical problems because of its low specific biological activity combined with high viscosity and poor solubility. Therefore, administering DS is cumbersome and impractical.
It has now been discovered that the biological activity of DS typically having one sulfate group per disaccharide may be significantly increased by the addition of another sulfate group. It also has been found that the resulting composition, dermatan disulfate (hereinafter DDS) consisting principally of repeating disulfated disaccharide residues not only is an effective thrombin inhibitor in vivo, but also effectively inhibits complement activation; and attenuates vessel wall hyperplasia. This is especially true for DDS consisting primarily of L-iduronic acid-&gt;4,6-di-O-sulfated-N-acetyl-D-galactosamine units (IdoA-GalNAc4S6S). Furthermore, it was found, that DDS inhibits thrombin generation more effectively than heparin during CPB.
As has been noted, DS comprises primarily monosulfated monomers, although some monomers in naturally occurring DS will be over-sulfated. Those DS polymers having a high L-iduronic acid content have been correlated with increased thrombin inhibition. (Whinna, H. C., Choi, H. U., Rosenberg, L. C., Church, F. C., Interaction of Heparin Cofactor II with Biglycan and Decorin, J. Biol. Chem. 268: 392S3924 (1993)) Moreover, it has been further suggested that increasing the degree of sulfation of DS improves the catalytic effects of inhibition of thrombin by HCII in plasma. (Ofosu, F. A., Modi, G. J., Smith, L. M., Cerskus, A. L., Hirsch, J., Blajchman, M. A. Blood 64: 742-47 (1984)) Other studies on slightly oversulfated derivatives of DS have suggested their potential as antithrombotic drugs. (Maaroufi, R. M., Tapon Bretaddiere, J., Mardiguian, J., Sternberg, C., Dautzenberg, M. D., Fischer, A. M,. Influence of the Oversulfation Method and the Degree of Sulfation on the Anticoagulant Properties of Dermatan Sulfate Derivatives, Thromb. Res. 59: 749-758 (1990)).
A linear correlation has been found in the content of disulfated disaccharide residues and the HCII mediated activity for dermatan sulfate fractions containing up to 20% of 2,4-O-disulfated disaccharides (IdoA2S-GalNAc4S). However, when the content of 2,4-O-disulfated disaccharides residues was low, even considerable concentrations of 4,6-O-disulfated disaccharides residues (IdoA-GalNAc4S6S) failed to contribute to antithrombin activity. (Mascellani, G., Liverani, L., Prete, A., Bergonzini, G., Bianchini, P., Torri, G., Bisio, A., Guerrini, M, and Casu, B., Quantitation of Dermatan. Sulfate Active Site for Heparin Cofactor II by H-Nuclear Magnetic Resonance Spectroscopy, Anal. Biochem. 223: 135-141 (1994)) Other studies attribute a high HCII-mediated inhibition of thrombin generation principally to dermatan sulfate fractions containing about 3% of 4,6-O-disulfated disaccharide sequences. (Linhardt, R. J., Desai, U. R., Liu, J., Pervin, A., Hoppensteadt, D., Fareed, J., Low Molecular Weight Dermatan Sulfate as an antithrombin agent, Biochem. Phanncol. 47: 1241-1252 (1994)) More recently, it was suggested that 4-O-sulfation of the N-acetyl-D-galactosamine residues is essential for the anticoagulant activity of DS and that the structure which binds to HCII is repeating 4-O-sulfated-L-iduronic-&gt;4-O-sulfated-D-galactosamine sequences. (Pavao, M. S. G., Mourao, P. A. S., Mulloy, B., Tollefsen, D. M., J. Biol. Chem. 270: 31027-36 (1995)).
Bovine mucosa and pig skin are a primary source for commercial dermatan sulfates preparations containing 2,4-O-disulfated disaccharide residues. (Mascellani, G., Liverani, L., Prete, A., Bergonzini, G., Bianchini, P., Torri, G., Bisio, A., Guerrini, M, and Casu, B., Quantitation of Dermatan. Sulfate Active Site for Heparin Cofactor II by H-Nuclear Magnetic Resonance Spectroscopy, Anal. Biochem. 223: 135-141 (1994)) Porcine mucosa derived dermatan sulfates typically have 4,6-O-disulfated disaccharide residues as well. (Mascellani, G., Liverani, L., Prete, A., Bergonzini, G., Bianchini, P., Torri, G., Bisio, A., Guerrini, M, and Casu, B. Anal. Biochem. 223: 135-141 (1994); Linhardt, R. J., Desai, U. R., Liu, J., Pervin, A., Hoppensteadt, D., Fareed, J., Low Molecular Weight Dermatan Sulfate as an antithrombin agent, Biochem. Phanncol. 47: 1241-1252 (1994)) A GAG from squid cartilage, also known as Chondroitin Sulfate E, is composed principally of 4,6-O-disulfated disaccharides. However, it is composed principally of D-glucuronic-&gt;4,6-O-disulfated-N-acetyl-D-galactosamine units (Kawai, Y., Seno, N., Anno, K., J. Biochem. 60: 317 (1966)) rather than L-iduronic-&gt;4,6-O-disulfated-N-acetyl-D-galactosamine (IdoA-GalNAc4S6S).
The degree of sulfation is an important functional property that contributes significantly to the anticoagulant effect of DS and other heparin-like GAGs. In DS, the galactosamine unit is typically O-sulfated and O-sulfate groups are often present at the 4-position and occasionally at the 6-position. The 2- and/or 3-position of iduronic acid are occasionally sulfated, as well.